The long-term aim of this project is to advance BILT for clinical approval which will provide clinicians with a powerful addition to the armamentarium for treating CTCL and cancer immunotherapy through depleting CD25+ Treg. Ontak(R) (denileukin diftitox) was making great strides in the treatment of CTCL since its FDA approval in 1999 until its discontinuation. BILT functions analogously to Ontak(R) but with greater potency. The functional parallel between these two therapies provides a clear pre-clinical and clinical path forward. An ideal cancer therapy would be effective, selective, permanent, and safe. The immune system has the ability to specifically recognize and attack tumor cells and their supportive microenvironments, while sparing nearby normal cells. We have developed a novel therapy that has met these marks through the use of a genetically engineered bivalent human IL-2 fused to a truncated diphtheria toxin (BILT). In addition, we developed a robust, scalable and efficient yeast expression system with greatly enhanced purification yields. Although Ontak(R) was an integral part of the treatment regimen for patients with CTCL, its discontinuation due to a supply interruption has introduced a gap. The work proposed here is a major step toward advancing a next-generation therapy that targets CD25+ tumor cells with greater potency than Ontak(R). In murine models, the bivalent fusion toxin was efficacious against CD25+ tumors and effectively depleted CD25+ tumor cells in vivo, while exhibiting no overt toxicity. BILT has the potential for significant efficacy against human disease, safety profile similar to that of an approved product, and improve the clinicians' toolkit against CTCL. Beyond its initial effect on CTCL, BILT is likely to efficacious against other CD25+ tumors, and may also impact organ transplantation where targeting CD25+ T cells is a key therapeutic goal. In this Phase I SBIR proposal, we will determine the anti-tumor efficacy of BILT in vivo, we will determine the maximum tolerated dose (MTD) and the mechanism of toxicity, and we will develop a fermentation SOP to support clinical development. These studies will advance BILT and, if successful, will position this therapy for large-animal preclinical and subsequent clinical development. At the end of this Phase I project, we will have achieved key goals on the critical path to move this promissing intervention toward the clinic.